Molecular Diagnostics of Corn Cyst, Heterodera zeae and Pea Cyst, Heterodera goettingiana Nematodes
abridged from: Szalanski, A.L., D.D. Sui, T.S. Harris, and T.O. Powers. 1997. Identification of cyst nematodes of agronomic and regulatory concern with PCR-RFLP of ITS1. Journal of Nematology 29: 255-267.

Amplified DNA was purified using Geneclean II (Bio 101, Inc. Vista, CA) following manufacturer protocols and resuspended in 30 ml of TE (pH 7.5). DNA was blunt ended using New England Biolabs (Beverly, MA) reagents and ligated using Stratagene (La Jolla, CA) reagents into pBluescript sk+ plasmid (Stratagene, La Jolla, CA) following Sambrook et al. (1989). Plasmid transformation and screening of clones were conducted following Sambrook et al. (1989). Positive colonies were verified by picking colonies with a sterile toothpick, replating the colony on a new LB ampicillin/tetracycline plate (Sambrook et al. 1989) and mixing the toothpick into a 50 ml PCR reaction mixture. PCR reaction was performed as before but for only 20 cycles. Five ml of PCR product was run on an agarose gel to determine if the insert was present. Single stranded product was obtained from positive clones and sequenced using a LI-COR Model 4000 DNA Sequencer (LI-COR Inc., Lincoln, NE) by the University of Nebraska-Lincoln DNA Sequencing Lab (Lincoln, NE). Two primers, T3 promotor and T7 promotor (Gibco BRL, Gaitherburg, MD), were used for sequencing in both directions. Sequences were aligned using GCG (Genetics Computer Group) PILEUP program (with a gapweight of 5.0 and a gaplengthweight of 0.3).  PCR-RFLP: Restriction sites were predicted from sequence data using the program Webcutter.  Sample DNA was amplified as previously described. Predicted diagnostic markers were checked with restriction enzymes from New England Biolabs (Beverly, MA). Amplified ITS1 DNA was digested according to manufacturer’s recommendations (New England Biolabs, Beverly, MA). The digestion reaction included 7.0 µl sterile double distilled H2O, 10.0 µl amplified DNA, 2.0 µl 10x restriction enzyme buffer, and 1.0 µl of restriction enzyme. Reactions were incubated at 37 °C for 6 - 24 hours. For electrophoresis of digested DNA, the digestion mixture was added to 1.6 µl of gel dye and loaded on a 2.5% MetaPhor (FMC, Rockland, ME) agarose gel in 0.5x Tris:Borate:EDTA (TBE) buffer on a model H5 horizontal gel apparatus (BRL, Gaithersburg, MD). Ethidium bromide (0.015 mg) was added to the gel and electrophoresis buffer. Electrophoresis was at a constant voltage of 100 V for 4 hours. Gels were photographed under UV light using Polaroid film. Prints were scanned using a HPIIcx flatbed scanner and Aldus photostyler software. 


The ITS1 primer set amplified a single fragment from both cyst DNA extractions and single egg or juvenile squashes.  The ITS1 amplification product was 798 bp in length (including primers) for H. zeae, and 811 bp in length for H. goettingiana as determined by nucleotide sequencing.  The restriction enzymes Alu I, Hha I, and Hinf I readily distinguished pea cyst nematode from corn cyst nematode (Fig. 1). Corn cyst nematode displayed several intraspecific restriction site differences when all five isolates were compared. For example, AvaI digestions of the U.S.A isolates produce a three fragment pattern compared to two fragment pattern observed among isolates from India. (Fig. 2). The AluI restriction pattern of the U.S.A. isolates display a 530 bp fragment not observed in the India isolates, and HaeIII digestion also produces two fragments, 424 and 118 bp, not seen in the Indian isolate (Fig. 2).